Mutation Screening of BRCA Genes in 10 Iranian Males with Breast Cancer.

Male breast cancer is a rare disease with an increasing trend. Due to limited information especially about the genetic basis of the disease in Iran and the lower age of its onset, the disease requires more attention. The aim of this study was to screen the male patients with breast cancer for BRCA mutations as well as tissue markers of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER-2) and cytokeratin 5/6 (CK5/6). Ten Iranian males with breast cancer were selected regardless of their histologic subtypes, age and family history from patients referred to Mehrad, Day and Parsian hospitals in Tehran, Iran, during a two-year period. Paraffin blocks of the tumoral regions were tested for ER, PR, HER-2 and CK5/6 immunostaining. DNA extraction was carried out on the EDTA blood samples followed by Sanger sequencing. Immunohistochemistry results for ER, and PR were negative in 2 out of 10 patients, while the results of HER-2 and CK5/6 were negative in all the cases. A missense mutation in exon 18 of BRCA1 and a nonsense mutation in exon 25 of in BRCA2 were detected in one patient each. Both patients belonged to luminal A subtype. Despite the low number of patients in this study, it could be concluded that mutations in BRCA1 and BRCA2 occur in male breast cancer patients of luminal A subtype. The negative status of the tissue markers could not be used for the prediction of BRCA mutations.

reast cancer is the most prevalent malignancy affecting women (1,2). The disease is rare in males, accounting for 1% of all breast cancer cases (3)(4)(5) but recently a significant increasing rate from 0.86 to 1.08 cases per 100,000 populations has been reported (5)(6)(7). There are many studies reported from Iran on female breast cancer (FBC) but male breast cancer (MBC) still needs to be studied (8,9).  (10). In Iran, MBC accounts for 0.65% of all cases of malignancy in men. There are 6674 incident cases of breast cancer diagnosed in Iran in 2007 of whom 3.26% were men (11). Due to its low incidence, there are few studies and limited information on this subject especially about the genetic etiology of the disease.
Furthermore, the average age of male breast cancer and female breast cancer in Iran is lower than developed countries (11), and men are usually diagnosed with breast cancer at more advanced stages often with lymph node metastasis (3,12).
Several risk factors including advancing age, positive family history and mutations in susceptibility genes such as BRCA1 and especially BRCA2 are considered for the study of male breast cancer (10).
BRCA1 and BRCA2 are tumor suppressor genes, located on 17q21and 13q12 loci with 24 and 27 exons, respectively. These genes normally participate in the repair of damaged DNA by homologous recombination. Although BRCA2 is the most clearly associated gene, BRCA1 mutation is also considered in familial and even sporadic cases of male breast cancer (4,5,13). According to some studies, BRCA2 mutations occur in 4-16% of male breast cancer patients. However, BRCA1 mutations are much less common and 0-4% of men with breast cancer carry these mutations (14).
BRCA1 mutations as a prognostic factor also have great importance, as carriers of these mutations have a poorer prognosis compared to other patients.
Mutation in BRCA2 also has a special significance as the mutation carriers can be diagnosed with breast cancer at lower age and has a shorter survival (13). In comparison with BRCA1 mutations, BRCA2 mutations have major significance in MBC patients (4,5,13). Patients with mutation in BRCA2 are predisposed to breast cancer and other malignancies such as pancreatic, prostate cancer and melanoma. Molecular subtypes of MBC are similar to FBC (4,11). According to several studies, MBC seems to be more hormone receptor positive (luminal A subtype) (3,13,14), than FBC. The main aim of this study was to screen the male patients with breast cancer for BRCA1 and BRCA2 mutations as well as the status of tissue tumor markers including ER, PR, HER-2 and basal marker (CK5/6). We also investigated the relationship between the BRCA1 and BRCA2 mutations and the tissue markers' status.

Patient selection and sampling
In this cross-sectional study, 10   Interpretation of the results was performed using the classification method based on tissue tumor markers (Table 1).

Mutation analysis
Following PCR amplification and sequencing of BRCA1 and BRCA2, in 10 males with breast cancer, their variants and polymorphisms were detected. Further information, including the exon number, base changes and frequency of these variants in 10 patients, are shown in Tables 2 and 3 for the BRCA1 and BRCA2, respectively. a b    of BRCA1 at position c.5158C>T (Figure 4a, Table   4). This mutation was evaluated by Mutation   Table 5.

Discussion
In this study, 10 males with breast cancer were screened for mutations in BRCA1 and BRCA2.   (18). While this project is a pioneer study based on genetic and histopathologic aspects of MBC in Iran, there is still a great need to study the genetic basis of MBC.
It should be mentioned that the main problems of this project were: "case finding" and "tiny tissue samples". Considering the low prevalence of the disease around the world, especially in Iran, despite searching four hospitals to find males with breast cancer, and due to the problems such as lack of cooperation of the patients and their families, finally a small population of only 10 qualified cases were allocated for the study. According to the rarity of MBC and its increasing rate in the world, it seems to be necessary to perform studies with larger sample size. We believe that general education for the early diagnosis of breast cancer in males in order to increase their awareness of this disease, and also promotion of the quality of diagnostic methods in Iran are necessary.